CN1212385C - Solid state fermentation tank and solid state fermentation process - Google Patents

Abstract

The present invention discloses a large-capacity solid-state fermentation tank and a solid-state fermentation process, so that solid-state fermentation can be economically performed in a large fermentation tank for microorganisms having very weak competitiveness. The invention relates to a solid-state fermenter module, characterized in that the solid-state fermenter module comprises at least two module bases which are permeable to air and water and are arranged in a stack, the module bases being connected to the wall of the tank and being impermeable to air and water from the sides; the culture medium of the microorganisms to be cultivated is placed on the module base; a cooling device is arranged under each module base, and the fermenter is closed with a lid.

Description

Solid state fermentation tank and solid state fermentation process

technical field

The invention relates to a solid-state fermentation tank, especially a large-capacity solid-state fermentation tank and a solid-state fermentation process.

Background technique

Submerged fermentation or solid-state fermentation is often used for the cultivation of large numbers of microorganisms, the purpose of which is to isolate the microorganisms themselves or metabolites or a medium that is modified by microorganisms (for example: in the food processing industry). At present, submerged fermenters (fermenters with liquid nutrient medium) with a capacity of up to 200.000 liters have been produced, but they have a certain capacity, can not be polluted by foreign microorganisms for a long period of time, and have optimal culture conditions at the same time. Solid-state fermenters (fermentors with solid nutrient media) have yet to be produced. However, the cultivation of certain filamentous molds requires surface structures that allow them to grow and form spores. The largest fermenter for the production of filamentous molds free from any external contamination was installed at the French INRA (Durand 1997, verbalcommunication), with a capacity of 50 liters, however, the capacity of this fermenter is not sufficient for the production of mold spores that can be used, for example, as biopesticides. It is not enough to produce economically.

Solid-state fermentation (SSF) is defined as the growth of microorganisms, usually molds, on solid-state media in a defined gaseous phase and in the absence of a free aqueous phase. In ancient times, some areas of the Far East, Asia, and Africa have already used solid-state fermentation to produce fermented food, enzyme products (Koji) or edible fungi. Western countries have focused on the research of submerged fermentation since 1940. Solid state fermentation is only used for reprocessing of organic waste. Recently, however, many institutions and manufacturers have expressed great interest in solid-state fermentation because of its many advantages over immersion fermentation. The advantages of solid-state fermentation over submerged fermentation are:

- Efficient production of secondary metabolites such as: enzymes, flavoring, aromatic and coloring substances as well as pharmaceutical actives;

- Microorganisms capable of producing biological agents in pesticides;

- Remove toxins or other harmful substances from food and feed or concentrate protein or vitamins in feed.

Basically, there are six types of solid state fermenters:

1. Disc bioreactor;

2. Packed bed bioreactor;

3. Rotating drum bioreactor;

4. Swing solid-state bioreactor;

5. Stirred container bioreactor;

6. Air solid fluidized bed bioreactor.

The first type - tray bioreactors, in which the fermented medium is spread flat in a vessel specially made for the purpose, for which the reactor is grown in an air-conditioned room ('Koji'-Raum, Ramana Murthy, MV; Karanth, NG; Raghava Rao, KSMS: Advance in Applied Microbiology 38 (1993), 99-147), disc bioreactors can be used for the production of large quantities of products, however it must be possible to avoid the Contamination from foreign pathogens. In addition, the reactor and the reaction method require a lot of space and a lot of labor. The fermented medium within the vessel must be manually moved. This method is not suitable for the mass production of very weak mold spores.

In a 'packed bed bioreactor', the moist granular medium used to grow the microorganisms is placed in a closed container where the microorganisms grow without moving the medium. For this reason, the culture medium has to be frequently aerated with air, so that the following problems arise, and a large amount of the culture medium cannot always be used.

1. Microbial heat production (300 kJ/kg (dry weight) hour, Saucedo-Castaneda, G.; Gutierrez-Rojas, M.; Bacquet, G.; Raimbault, M.; Viniegra-Gonzalez, G.; Biotechnologie and Bioengeniering 35 (1990), 802-808), the heat can be diffused either by the outer wall of the container or by increasing air circulation (evaporative cooling). However, if it is a large-capacity container, the above-mentioned heat dissipation is impossible. The growth rate of microorganisms will slow down with the increase of heat until finally necrosis.

2. Frequent aeration to dry the culture medium will cause 'water loss', resulting in stomata. The presence of stomata can no longer ensure uniform aeration of the culture medium. Gradual drying of the medium can in turn lead to deterioration of microbial growth.

A 'rotating drum bioreactor' consists of a horizontally mounted cylindrical vessel that rotates on a pivot. Only one-third of the vessel's capacity is filled with granular medium for microbial growth. Most of the heat generated by the microbial growth is evaporated by the locally cooled container shell. As the cylindrical container rotates slowly, the medium comes into contact with the enclosure again and again, releasing heat to the enclosure. But this method also has disadvantages, in the process of moving the medium, the shear force acts, especially the mold structure (mycelium, sporangia, fruiting body) in the growth process will be destroyed. In this way it has not always been possible for many molds to obtain high yields of spores. In this type of fermenter, the solution to the dryness problem is basically to introduce humid air, since there is no need to evaporate water from the medium (no evaporative cooling required). In addition, the nozzles can achieve the wetting of the medium and also allow a good distribution of free water through movement.

However, growing large volumes of media in such fermenters poses other problems:

1. Design a large fermentation tank, which is very expensive;

2. The continuous movement of the fermenter will cause the agglomeration of the wet medium;

3. It is necessary to have interfaces leading to the outside world (air inlet and outlet, water inlet). During the rotation process of the fermenter, they are easy to become the source of foreign germ pollution.

A type of fermenter similar to the 'rotating drum bioreactor' is the 'swinging solid state bioreactor', the only difference being that the medium is mixed not by rotational motion but by oscillatory motion. The advantages and disadvantages of this reactor are the same as those of the drum bioreactor mentioned above. However, another reason why the capacity of this fermenter is limited is that the structure of the oscillating mechanism is complicated, which does not allow the weight of the container containing the medium to exceed 100 kg.

A 'stirred vessel bioreactor' can be described as a closed tank with a moving agitator inside. The use of a large amount of medium in such a reactor inevitably creates problems because it is impossible to uniformly move such a large amount of medium without destroying the structure of the medium.

In the 'air-solid fluidized bed bioreactor', the medium for microbial growth is always placed in the fluidized bed, which necessitates a relatively large volume of reaction chamber. The air required to maintain the work of the fluidized bed is introduced by circulation. This air must be maintained at a precisely calculated humidity. The process of maintaining a fluidized bed in operation requires a lot of energy. The possibility of culturing yeast cells in a fluidized bed was demonstrated in the already implemented AiF project (Bahr, d.; Menner, M.; BIOforum 18 , 1995, 16-21).

However, compared to submerged fermentation, air-solids fluidized bed bioreactors are considerably smaller in size and lower in yield. Only under the premise of very high cost with this technology, it is possible to carry out a period of several weeks of filamentous mold cultivation on a large amount of granular medium (each batch greater than 100 kg), which is not advisable.

Other existing fermenters are also too small to use them to obtain economically applicable quantities of mold spores (EP-A1-0 683 815 and FR 85.08555), or fermenters of sufficient capacity to exclude foreign substances for a long period of time. Possibility of contamination of the culture medium by pathogens (DE 4406632C1).

Disclosure of the invention

Therefore, the object of the present invention is to provide a large-capacity solid-state fermenter and a solid-state fermentation process, so that solid-state fermentation can be economically applied to very weakly competitive microbial large-scale fermenters.

It must be:

1. Avoid external contamination of the fermenter (maintain sterile conditions throughout the fermentation process),

2. Release the heat generated by mold metabolism to avoid drying of the medium (by increasing air flow and evaporative cooling),

3. Avoidance of shear stress in the fermenter (medium does not move), and

4. Ensure uniform aeration (avoid drying) and control the temperature of the medium.

The invention is realized according to the claims. The object of the invention is solved with a modular fermenter. The fermenter has a capacity of at least 50 liters, preferably 500-1000 liters, but can be larger. The whole structure consists of a cylindrical or elliptical cylindrical container (as shown in Figure 1), which can be closed from above by a cover 1. If necessary, an air outlet 2 and a place for inoculation of the fermenter can also be provided on the cover. The small hole used 3.

The container is made as an air- and water-tight shell comprising support plate modules 4 stacked one above the other and permeable to air and vapour. The module is stacked with a culture medium 5 for growing microorganisms.

The culture medium is composed of different substances according to the respective nutritional requirements of the microorganisms to be cultured. These materials preferably have a granular structure to ensure adequate air penetration. For example: The substances that make up the culture medium can be grains, bran grains or other organic wastes, wastes from the sugar production process or granules soaked in nutrient solution.

The number of layers depends on the culture needs of the microorganisms being cultured and the ease of maintenance of the entire fermenter. Too many layers will affect the oxygen supply necessary for the growth of the microorganisms on the upper layer of the medium (see below for details). Moreover, too many layers are not conducive to the maintenance of the fermenter. Nevertheless, according to the present invention, the fermenter will be installed with 20 floors or more.

The support plate module is connected to the container wall in such a way that neither air nor water can flow through the side of the module. The distance between the support plate modules depends on the optimal layer thickness of the medium and, on the other hand, on the requirements of the microorganisms to be cultivated.

A cooling device 6 is installed below the support plate module, which can be made into a cooling serpentine tube or a cooling fin to release the heat generated by the reaction of the culture medium. In a proposed solution, metal sheets with high thermal conductivity can pass from each cooling unit through a support plate module into the culture medium (as shown in Figure 2). In this way, the heat of reaction is released more easily. After the fermentation process is complete, the cooling unit is pulled out from under the support plate module together with the cooling fins to move the culture medium. Next, the medium with the mature microorganisms can be removed without the cooling fins getting in the way.

It is also possible to install the cooling device within a certain distance on the support plate module. In this case, the cooling device works in the middle of the medium layer. The cooling device is installed in the middle of the culture medium layer and parallel to the support plate module, especially suitable for the situation of high heat generation during the fermentation process.

The base of the fermenter has an air inlet 7 through which sterile, moist air is blown into the fermenter. After the air flows through each layer of medium, it is exhausted from the air outlet 2 located on the lid of the fermenter.

The spaces between the modules are also fitted with cooling devices to ensure an even distribution of air throughout the fermenter. The air in the fermenter can also be humidified if there is no humid air for aeration in the fermenter, this is done by filling at least the lowest support plate modules not with culture medium but with granular material, The material absorbs moisture so that the blown air first flows through these materials, humidifying the air before penetrating into the fermenter. If large amounts of moisture are required for microbial growth, a plurality of such air-humidifying modules can be installed at a distance in the fermenter. The amount of air blown depends on the oxygen demand of the microorganisms being cultivated. It can range between 1 and 100 liters per hour per liter of medium.

The sterile water injected into the fermenter should reach the top layer of the medium for the inoculation of the sterilized medium with the cultured microorganisms. For this purpose, a water inlet equipped with a sterilizing filter is provided. 8. The water inlet can also be located in other positions of the fermenter (for example: on the lid). After the water injection, the culture solution is added through a dedicated small hole 3 located on the lid. Such small holes 3 for the inoculation of the fermenter can also be provided between individual support plate modules, especially if a number of support plate modules are provided. In the above first case, the distribution of the culture solution in the fermenter can only be realized by discharging water through the special water outlet 9 located at the bottom of the fermenter.

The broth (suspension of microorganisms) flows through the layers of the medium in such a way that a sufficient amount of broth with added water adheres to the medium. If a large number of layers are required, depending on the structure of the medium, a dilution effect will occur, that is, microorganisms will be filtered as they pass through the medium, so that their concentration in the water will be lower than the actual concentration. In order to prevent this phenomenon from happening, in another solution, small holes for injecting culture solution into the fermenter can also be arranged between the support plate modules. In the process of injecting water into the fermenter, the culture solution may have been injected, and then the culture solution is distributed with the water flow, the direction of the water flow is upward, the culture solution flows upward, and the direction of the water flow is downward, the culture solution flows downward.

Broth for fermentor fermentations consisting of a highly concentrated suspension of small primordial units (preferably spores, conidiophores or bacterial germs) of the microorganisms being cultivated.

When the inoculation container has a uniform and sufficient inoculation condition, the cultivation process (cultivation period and product yield) and the quality of the culture product (such as mold spores) mainly depend on various factors in the cultivation process. The main factors are the introduction of humid air and temperature control. The air volumetric flow rate should be adapted to the capacity of the air sterilization filter. The temperature control in the fermenter is guaranteed by the cooling device installed in the tank, and its cooling capacity should be designed to release all the heat generated by the reaction of the medium and maintain an optimal temperature for cultivating microorganisms. The required cooling capacity also depends on the layer thickness and volume of the medium. The more medium used for microbial growth, the more heat generated by the reaction. This is why both factors must be optimal. The goal is to develop the microorganisms as fast as possible and to produce as high a product as possible. Depending on the purpose of the fermentation, the products can be mold spores, bacterial cells, enzymes, antibiotics, coloring materials and other substances.

According to the present invention, the following two fermenter designs are provided.

Option 1 (see Figure 3)

The fermenter consists of a delicate round or prismatic cylinder with a closed bottom. Cylindrical (usually circular) or prismatic cylinders with a diameter of 1 m or more, the height of which is limited by two factors, namely technical ease of maintenance and the possibility of ensuring optimum conditions for the growth of microorganisms . Possible heights are 2 meters or more.

The support plate module 4 with the growth medium 5 is inserted into the circular or prismatic cylinder from above. Inside the container there are rings and, if a prismatic cylinder is used, members 11 of different shape to support the support plate modules. Each ring or support member of different shape is equipped with a heat-resistant gasket 10, for example: silicone gasket, on which the outer edge of the support plate module is placed, so that the space between the support plate module and the container wall can be fixed. Sealed to prevent air and water from penetrating. Rings or differently shaped support members can also be removed from the cartridge. The cooling device 6 located under the support plate module can be composed of, for example, a copper cooling serpentine pipe, which is connected to the conduits 14 installed outside the fermenter and leading to the cooling liquid inlet and outlet respectively through a quick connector 13 . Each support plate module has an edge 12 whose height is adjusted according to the layer thickness of the culture medium. In this way, it is possible to avoid contamination by medium falling into the fermenter container.

The top of the fermenter is tightly closed by a lid 1 . The fermenter is designed like a pressure vessel and can also be sterilized by sending hot steam under pressure, so there is no need to use an autoclave.

Option 2 (see Figure 4)

The fermenter consists of several short (preferably about 7-30 cm in height) circular or prismatic cylinders, so that the support plate modules can be annular, oval, rectangular or other angular. In each case, each individual cylinder or prismatic cylinder is fitted with an air- and water-permeable bottom. The cooling device is located below this bottom, above which is the culture medium for cultivating microorganisms. For the composite fermenter, the circular tube or the prismatic tube is used as the module 4. They are stacked on top of each other and sealed against each other by heat-resistant gaskets 15 located at the edge of the cylinder. The first module is placed horizontally against the bottom of the fermenter, the last module is on top and is closed by the lid of the fermenter. Thus, the fermenter preferably consists of 10 or more such modules. Because it is difficult to design such a composite fermenter as a pressure vessel, the sterilization of the fermenter and the culture medium in the tank is carried out in an autoclave. Thus, the height of the fermenter depends primarily on the capacity of the autoclave used. Therefore, in most cases, the volume of the fermenter will be limited to 500-1000 liters. During the autoclaving process, the fermenter is still open, that is to say, the modules are lifted slightly (approximately 5 mm) from each other, so that hot steam can enter the interior of the fermenter for sterilization. After autoclaving, the fermenters are tightly closed. Each module is fitted with an outer ring 16 for covering the gaps that exist between the modules, to avoid when the fermenter is opened, i.e. after autoclaving, and before the fermenter is closed, i.e. when the fermenter has just come out of the autoclave. When the medium is taken out, the contamination of the fermenter by foreign pathogens.

When the fermenter is closed, the cooling device 6 located under the support plate modules is connected via a connection 17 to a conduit 14 for the supply and discharge of cooling liquid.

In the recommended design, each layer of granular medium for microbial growth is 5-6 cm thick, and up to 10 such layers are stacked on top of each other. Each layer of granular media rests on a porous bottom that is permeable to air. Under the bottom surface is a cooling coil (copper tube can be used) to release the heat generated by the medium. Sterile, filtered air is supplied from the bottom. As the sides are closed, the air is forced to circulate evenly between the modules (media layers) before it is exhausted again from the top of the fermenter. The lowest support board module is the water saturated layer, preferably SERAMIS granules, so that the air can be moistened when passing through it.

The fermenter and the medium already placed in the fermenter, preferably in an autoclave, are heated to 121°C and steam sterilized, during which the individual modules are lifted slightly away from each other so that hot steam can enter the modules .

Other data:

Volume 500 liters

                                                                               

Air volume flow 1500 liters/hour

    Cooling System Power                                                        

Different from the currently used fermentors (swinging solid-state fermenters or drum fermenters), the currently used fermentors realize heat loss, ventilation and water supply by constantly turning over the culture medium, and according to the present invention With the provided method, it is unnecessary to move the culture medium. The culture medium is placed layer by layer, and is packaged as a whole by a closed shell, which has the following advantages:

1. The weight of the medium itself will not cause compaction, so this placement will not reduce the air permeability.

2. A cooling device is installed under a single module, so that the heat generated by the medium can be easily dissipated.

3. Since the thickness of each module is relatively small, and the gap between the modules is also relatively small, the uniform ventilation of the culture medium layer can be ensured.

4. Since the aeration of the culture medium is only used to supply oxygen and discharge the generated gas, it is not used to cool the culture medium, so it can work with a very low air volume flow, and because the air is humid, it will not cause cultivation The base is dry.

5. Since there is no need to move the medium, mechanical damage to mold structures (spore sacs, fruiting bodies, etc.) can be avoided.

Brief description of the drawings

Fig. 1 is the schematic diagram of fermentation tank of the present invention;

Fig. 2 is the schematic diagram of the cooling device of the fermenter with heat-conducting sheet of the present invention;

Fig. 3 is the sectional view of the fermentation tank that the present invention is made of a circular cylinder;

Fig. 4 is a sectional view of an assembled fermenter of the present invention.

Best Mode for Carrying Out the Invention

Example 1

Mass cultivation of Beauveria bassiana brongniartii for the production of fungal conidia

The capacity of the fermenter for cultivating Beauveria bassiana brongniartii is about 50 liters, and its shape is a circular cylinder with a diameter of 30 cm and a height of 70 cm. The shell of the fermenter is made of heat-resistant glass. Eight modules are installed in the fermenter, and the bottom of each module is made of a stainless steel screen with a pore size of 3 mm. The distance between the support plate modules is 8 cm and the bottom layer is filled with a 6 cm layer of SERAMIS granules. The top seven modules contain crushed barley kernels used as culture medium. The thickness of each layer of medium is about 6 cm. A total of 30 liters of medium was used.

The fermenter is sterilized in an autoclave, and the fermenter is heated to 121°C with hot steam and sterilized for half an hour. During the sterilization process, the lid of the fermenter is opened slightly to allow hot steam to enter the interior of the fermenter. Close the lid immediately after disinfection.

Fill the fermenter with sterile water for inoculation, covering the top layer of the culture medium. A 500 cm2 container of the type S+S-EXELON PES 20/5 HC (Schleicher und Schuell, Dassel) was used for infusion of sterile water. Then, the culture solution is injected through a dedicated small hole located in the lid. The inoculation of the fermenter takes place inside a layered box. The culture medium used was 100 ml of conidia suspension containing 1 x 10 ⁹ conidia/ml. After the broth is injected into the top layer of the medium, the water is drained from a valve located at the bottom of the fermenter. In this way, each layer of culture medium is uniformly contaminated with fungal conidia.

After the fermenter is inoculated, it will be grown in a room at a temperature of 20 °C. At this point, it will be about the air supply and cooling system. The volume flow of air was 150 l/h throughout the fermentation. Water at a temperature of 17°C was used as cooling liquid. Control of cooling is regulated in such a way that cooling liquid is pumped through the cooling coil, if the temperature of the culture medium exceeds 22°C, until it is cooled down to 20°C again. In this way, the temperature of the culture medium can be maintained at an average of around 21°C throughout the culture process.

The goal of breeding is to produce as many mold conidia as possible. The whole fermentation process can be well observed through the glass shell of the fermenter. After about 10 days, the whole medium was covered with a layer of white mycelium. From the 13th day, the mycelium changed its appearance and became a powdery structure due to the formation of conidia and conidiophores. After about 19 days, metabolic activity was significantly reduced. As heat is reduced, cooling frequency is significantly reduced. The culture medium was removed 21 days after the inoculation of the fermenter, the conidia were extracted from the culture medium by a special filtration technique, and then the conidia were fully grown into B. bassiana brongniartii. A total of 3.3×10 ¹³ conidia were extracted from the fermenter module.

Explanation of reference signs

1 cover

2 Air outlet

3 Inoculation holes

4 Air-permeable support plate modules

5 culture medium

6 cooling device

7 Air inlet

8 water inlet

9 Drain outlet

10 Heat-resistant washer

11 Members used to support the support plate module

12 Edge of support plate module

13 Quick connector

14 Conduit for supply and discharge of cooling liquid

15 Heat-resistant washer

16 outer ring

17 connector

Claims (16)

1. A solid fermenter, characterized in that the fermenter module at least includes two support plate modules (4) that can permeate air and water and stack up; the support plate modules are connected to the container wall , neither air nor water can pass through from the side, A culture medium (5) for cultivating microorganisms is placed on the support plate module (4), Cooling devices (6) are installed below each support plate module (4) and the fermenter is closed with a lid (1). 2. The fermenter according to claim 1, characterized in that there is a container of a delicate circular cylinder, oval cylinder, rectangular cylinder or various angular cylinders, and the container has at least one small hole for inoculation (3). 3. The fermenter according to claim 1 or 2, characterized in that a metal sheet with high thermal conductivity protrudes from the cooling device and enters the culture medium through each support plate module. 4. The fermenter according to claim 1, characterized in that a water inlet (8) is arranged on the container, an air inlet (7) and a drain (9) are arranged at the bottom of the fermenter, and the lid (1 ) has an air outlet (2); The support plate modules (4) are stacked one on top of the other, and the culture medium (5) is filled on it, and ring-shaped or differently shaped members (11) are installed inside the container to support the support plate modules (4). The heat washer (10), the support plate module has an edge (12), the height of which depends on the thickness of the medium and the cooling device (6), which is connected via a quick coupling (13) to the Conduits (14) for the supply and discharge of cooling liquid are connected outside the fermenter. 5. The fermenter according to claim 4, characterized in that the position of the water inlet (8) is located on the bottom or the cover of the fermenter. 6. Fermenter according to claim 2, characterized in that holes for inoculation are provided in the lid and, if desired, also small holes for inoculation can be provided between the individual support plate modules. 7. The fermenter according to claim 1, characterized in that the fermenter comprises several circular cylinders, oval cylinders or prismatic cylinder containers, and these containers or modules are stacked one by one, that is, the first one is Placed at the bottom of the fermenter, the last container is closed by a lid (1), and each container is closed with a heat-resistant gasket (15), the container has an outer ring (16), Each support plate module (4) can permeate air and water, the upper part of the support plate module is the culture medium (5), and the lower part is the cooling device (6). Conduits (14) for the supply and discharge of cooling liquid are connected. 8. The fermenter according to claim 1, characterized in that the culture medium is air-permeable granules added with nutrient solution or natural granular substances. 9. The fermenter according to claim 8, characterized in that said natural granular substances are grains, bran grains or wastes from sugar production. 10. The fermenter according to claim 1, characterized in that there is a wetting layer on at least the lowest support plate module. 11. The fermenter according to claim 10, characterized in that the wetting layer is a water-absorbing particulate material with extremely large pores. 12. Fermenter according to claim 1, characterized in that the cooling device (6) is mounted within a certain distance on the support plate module (4) and parallel to it. 13. The fermenter according to claim 12, characterized in that the cooling device (6) is installed in the middle of the medium layer. 14. The solid-state fermentation process carried out with the equipment of claim 1, characterized in that the culture medium on several support plate modules in the fermenter can be evenly inoculated, and can be completely cultured with a relatively low air flow The substrates are circulated and the optimal temperature is adjusted by the cooling system according to the respective cultivation process. 15. The fermentation process according to claim 14, characterized in that the inoculation of microorganisms is to inject a sufficient amount of microorganisms into the fermenter in a water injection manner, and in the process of water injection and drainage, the microorganisms evenly flow through each layer and inoculate. 16. The fermentation process according to claim 15, characterized in that the cooling capacity depends on the volume of the culture medium, so as to ensure that all the heat generated by the reaction is discharged from the culture medium.

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